Metabolic engineering is a powerful approach for harnessing the tremendous biosynthetic capability of microorganisms, including primary and secondary pathways. A key aspect of this overall approach is the identification and amplification of rate-limiting steps to enhance production of valuable compounds. Since microbial metabolism is dynamically controlled by cellular events relating to growth and differentiation, rational pathway manipulation must consider a complex set of variables. Thus, the current challenge is metabolic engineering is to develop a comprehensive strategy that examines the temporal and spatial profiles that affect microbial biosynthetic potential. The primary aim of the proposed research is to develop a rational approach for metabolic engineering of secondary metabolite production using the cephamycin C biosynthetic pathway in Streptomyces clavuligerus as the model system. In this work molecular genetic, physiological and mathematical modeling approaches will be combined to obtain information on the temporal and spatial expression of rate- limiting enzymes and dclX, a recently discovered positive regulatory gene for this important class of natural products. A new in-vivo reporter will be employed by coupling promoter sequences and structural genes to the gene encoding green fluorescent protein. Temporal and spatial gene expression patterns in S. clavuligerus will be assessed and quantified using confocal microscopy. In addition, a regulable promoter system will be used to control expression of dclX and structural genes encoding rate-limiting enzymes. This unique tool will provide a strategy to investigate the effect of perturbing temporal expression patterns of key enzymes in this important metabolic system. Furthermore, a mathematical model will be developed to predict the effect of temporal perturbation of the biosynthetic machinery on cephamycin C biosynthesis. Overall, this work should provide important insight in to the temporal and spatial relationships of gene expression and protein localization within the beta-lactam pathways in Streptomyces and other microorganisms. Moreover, it is hoped that this work will provide an important theoretical and experimental base for the rational manipulation of complex metabolic pathways for novel metabolite production and improved efficiency. Please visit: http://www.cems.umn.edu/~hu-grp/gfp/nih.html)